High Efficiency Yeast Transformation
'High-Efficiency Yeast Transformation' Note: a revised version of this protocol is at High Efficiency Transformation 2.0 This is the easiest high-efficiency method I’ve found (from C. Boone’s website). It’s ideal for integrating nutritional or drug-resistance markers as well as auxotrophy markers, in place of any genomic locus you’d care to delete. NOTE: BEFORE YOU START I SUGGEST YOU MAKE THE REAGENTS AT THE END (GOOP and CARRIER DNA) BEFORE YOU START-scott For each transformation... 1) Pellet yeast from 3 ml of a mid-log culture at 3000 rpm for 2 min in a 15ml conical tube in the tabletop fuge swinging bucket rotor. (see below for ideas on how to grow yeast). 2) Aspirate supernatant. Resuspend in 3 ml 0.1 M LiOAc (P/N: Alfa Aesar, A17921) and re-spin at 3000 rpm for 2 min in a 15ml conical tube in the tabletop fuge swinging bucket rotor. Mark's LiOAC large stocks are lower left shelf above bench; small aliquots in "Amino Acid Stocks" drawer. If you don't work with Mark make your own stocks. 3) Aspirate Supernatant. Resuspend in 1 ml 0.1 M LiOAc, transfer to a microfuge tube, and re-spin in a microfuge tube at 3000 rpm for 2 min in the microfuge. 4) Aspirate using P1000. Resuspend in 30 ul 0.1 M LiOAc + carrier DNA1. (In the microfuge tube). Carrier DNA is Salmon Sperm DNA (P/N: SIGMA D1626-5G, or Invitrogen 15632-011 )', SSDNA is in 1.5ml aliquots in a box in the bottom of -20C #2. Boil THE CARRIER DNA ONLY NOT YOUR YEAST SAMPLE!!!!!!!!! by placing into 95C heat block on Katie's bench. 5) Add up to 15 ul DNA (e.g., PCR product that has been purified using a Qiaquick column). 6) Sit 15 min. at room temp. 7) Add 150 ul Goop2. 8) Sit 30 min at RT. 9) Incubate in a 42 water bath for 15 min. 10) Spin yeast out ''slowly (2000 rpm, 2 min). 11) Aspirate supernatant and resuspend yeast in 0.5 ml YPD. 12) Leave at 30C (incubator, not shaker- just leave the tubes in a rack) for 1 hr (or 4 hr if the phenotypic lag of a drug-resistance marker needs to be overcome). 13) Gently re-spin at 2000 rpm for 2 min and resuspend in 500 ul of water. 14) Plate onto selective plate. When only one a couple of transformations per parent strain are being done, can transform in the morning since dilutions of an overnight YPD culture can be done when the overnight is started (e.g., 4 ml of innoculated YPD can be diluted 5x, 25x, 125x and 625x). In the morning, the culture that is closest to an OD of 0.5 to 2 is used and rest discarded. When doing more than a couple of transformations per parent strain, 25 ml or more of YPD in a standard 125 ml Wheaton bottle is innoculated with 2 ml late log overnight culture (make several dilutions of the 2 ml culture when it is started and discard the ones you don’t need in the morning). 1 CARRIER DNA: For each 10 transformations add 0.3 ml 0.1 M LiOAc to 30ul (freshly boiled and then cooled to ROOM TEMPERATURE) 10 mg/ml salmon sperm DNA. Again this comes from 1.5ml aliquots in box in bottom of -20C #2. 2 GOOP: For each 10 transformations freshly mix 0.4 ml 0.5 M LiOAC and 1.6 ml Boone PEG (Boone PEG: dissolve 150 gm PEG3550 in 165 ml ddwater and filter sterilize). PEG3550 is in chemical shelves. '''LiOAc (P/N: Alfa Aesar, A17921) Salmon Sperm DNA (P/N: SIGMA D1626-5G, or Invitrogen 15632-011) PEG 3550 (P/N: Sigma, 3640) TE BUFFER: RECIPE http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8018 TE CONTAINS: TRIS-HCl (P/N: Sigma, T3253-500G) EDTA (P/N: OmniPur, 4050) Dan Lockshon 1/11/07, modified mm 082112 Key to pRS Yeast Vectors